This study is engaged in detecting and characterizing oxygenated intermediates which occur in enzyme-catalyzed reactions. Reduced forms of several redox-active enzymes will react with molecular oxygen, and rapid reaction techniques, including stopped-flow optical spectrophotometry and rapid freeze-quenching combined with Mossbauer and EPR spectrometry, will be employed to examine possible labile intermediates. The enzymes to be studied are: liver microsomal P-450LM monooxygenase and erythrocyte formyl-containing heme protein as examples of hemoproteins; bacterial parahydroxybenzoate hydroxylase and bacterial flavodoxin as examples of flavoproteins; and protocatechuate dioxygenase as an example of a nonheme iron-containing protein. It is hoped that some general requirements and properties associated with the mechanisms for the biological activation of oxygen will be learned during the course of these studies. In addition to these studies, work utilizing the same techniques will be done on the flavin-disulfide enzyme, lipoyl dehydrogenase, to try and further understand the mechanism of enzymatic disulfide-sulfhydryl interchange. Many of the above studies will be performed in collaboration with the following investigators and their colleagues (all Department of Biological Chemistry): Drs. Vincent Massey, M.J. Coon, D.E. Hultquist, and R.G. Matthews.